Human JC polyomavirus (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a life-threatening demyelinating disease of the central nervous system (CNS) resulting from infection of oligodendrocytes. JCV infection is prevalent in the human population, where it persists lifelong as an asymptomatic infection in healthy individuals. JCV-induced PML manifests in individuals immunosuppressed by HIV/AIDS, leukemia, allograft transplantation, or, more recently, monoclonal antibody-mediated therapy for autoimmune and inflammatory diseases. Natalizumab, a monoclonal antibody therapy for treatment of multiple sclerosis, carries a risk for PML; incidence of PML in these patients rises with duration of treatment. This therapy is intended to prevent migration of autoimmune T cells to their target tissues, but blockade of trafficking of JCV-specific T cells into the CNS is generally considered its adverse consequence. JCV isolated from PML patients were found to have mutations in the major capsid protein VP1 and the non-coding control region (NCCR), implicating virus-associated risk factors in addition to host immune compromise. Several specific VP1 mutations have been identified, all of which are single amino acid substitutions that map to sialic acid binding sites, which are involved in attachment and entry of virus into host cells. Species-specificity of polyomaviruses has severely impeded development of a tractable JCV-PML animal model, and as a result our understanding of the pathogenesis of PML is limited. For example, it remains to be determined if mutations in VP1 alter virulence, and the role that immunosuppression plays in disease progression. The goal of this project is to develop a model of PML in mice using mouse polyomavirus (MPyV) to investigate how virus capsid mutations, adaptive immune system deficiencies, and treatment with natalizumab contribute to induction of polyomavirus- induced CNS disease. Introduction of a PML-associated substitution into an analogous site in VP1 of MPyV is hypothesized to increase the severity of CNS disease in mice by making MPyV neurotropic, allowing infection and damage of glial cells, including oligodendrocytes. Shifts in tropism will be determined by quantitative polymerase chain reaction (PCR) to assess viral replication efficiency, immunofluorescence microscopy to define infected CNS cells, and histopathologic evaluation of CNS injury. As in human PML, CNS infection is likely exacerbated by immunosuppression. Using therapy similar to natalizumab in an MPyV CNS infection model, flow cytometry-based phenotypic and functional analyses will be used to determine the mechanisms by which immunosuppression contributes to pathogenesis. Successful development of this MPyV infection model would provide the opportunity to explore mechanisms of polyomavirus CNS pathogenesis in a natural host.